DE4130986A1 - PINOSYLVIN SYNTHASE GENES - Google Patents

Description

The present invention relates to new plants isolated genes for pinosilvin synthase and their uses for the transformation of vectors, host organisms and plants and for the production of plants which have increased resistance to pests.

The 3,5-dihydroxy-stilbene is called Pinosylvin net, which occurs in plants and against damage succeed, especially fungi, bacteria and insects has a toxic effect and is therefore suitable for these pests ward off. The ability to synthesize these substances through the plants is used as an important defense mechanism viewed. Unfortunately, only a few crop plants have the fa ability to form Pinosylvin, or to an extent testify which gives them sufficient resistance to Gives pests.

The use of stilbene synthase genes for generation of plants with increased pest resistance already known from EP-A-03 09 862. In this release Publication is specifically a Resveratrolsynthase gene from peanut plants (Arachis hypogea).  

New genes for pinosylvine synthase ("Pino sylvinsynthase genes ") found in the genetic material (the genome) of plants that can be incorporated no Pinosylvin or insufficient Pinosylvin gene, which increases the resistance of these plants against pests.

Surprisingly, the new Pinosylvinsyn thase genes much cheaper pest resistance in plants than that of the prior art knew resveratrol synthase gene from peanut.

Every nucleic acid is said to be under pinosylvine synthase genes (DNA) can be understood after its transcription in RNA and translation in protein the formation of a Enzyme, which has the properties of a pino sylvinsynthase, which nucleic acid from isolated in their natural environment or in a vector is integrated or in a prokaryotic or eu caryotic DNA as "foreign" DNA or as "additional Liche "DNA is included.

Such pinos are also to be found among pinosylvin synthase genes sylvinsynthase genes are understood to be on their Beginning and / or end still contain DNA sequences that the function of the genes is not or not significantly impaired other. These DNA, also known as "gene units" Sequences arise, e.g. B. by cutting out with Restriction enzymes since there are no interfaces for common ones Restriction enzymes exactly at the beginning and at the end of the gene available. The Pinosylvinsynthase genes or the gene Ein  Such DNA sequences can also be found at their ends wear which are adapted for their handling (e.g. "Linker").

The Pinosylvinsynthase genes (or the gene units) can be in the form found in the genome of Plants are included ("genomic" form, a finally not coding for pinosylvine synthase and / or non-regulatory sequences (such as Introns)) or in a form that the cDNA ("copy" DNA) that corresponds to mRNA with the help of reverse Transcriptase / Polymerase is available (and no In trons contains more). The Pinosylvinsynthase genes can also in partially or completely synthetic form available. Among synthetic genes there are also genes understood which by the new assembly of Tei len natural genes arise.

In the pinosylvine synthase genes according to the invention (or the gene units) can DNA sections by in we significant equivalent sections of DNA or DNA’s to be replaced.

In the present context, "foreign" DNA, such DNA can be understood, which in a certain prokaryotic or eukaryotic genome not na occurs naturally, but only through interventions by the People is included in this genome. "Additional" DNA should be such DNA, which in the respective pro caryotic or eukaryotic genome, of course occurs, but in additional quantities through interventions  by being included in this genome by humans. The "Foreign" DNA or "additional" DNA can be used as needed and nature of the present case in one or more Copies can be installed.

Pinosylvinsynthase, which with the participation of the invent Pinosylvinsynthase genes according to the invention (or the gene Ein units) is formed in plants or plant cells, means any enzyme which is like pinosylvine synthase works and in plants their resistance to damage lingen increased.

The preferred pinosylvine synthase Ge according to the invention ne are characterized in that they with the Plas mid pin 5-49 contained cDNA sequence or its parts or with the cDNA sequence according to SEO ID No: 1 or their Share hybridize and code for Pinosylvinsynthase ren.

Preferred Pinosylvinsynthase genes according to the invention the Pinosylvinsynthase genes, which are found in Kiefer (Pinus sp.), particularly preferably in Pinus sylvestris and can be isolated from it.

Is very particularly preferred as the Pi according to the invention nosylvinsynthase gene the Pinosylvinsynthase gene, the Partial sequence in the form of the cDNA on the plasmid pin 5-49, (which is described in more detail below), as well as the essentially equivalent DNA sequences Zen.  

The cDNA contained on the plasmid was from Pinus sylvestris isolated. It consists of an approx. 1300 Base pair long sequence. A partial sequence of 570 Base pairs comes from the sequence listing SEO ID No: 1 forth.

It has been found that those found in plants (especially Pine and particularly preferably Pinus sylvestris) coming Pinosylvinsynthase genes over wide ranges have a DNA sequence homology. The fiction According to Pinosylvinsynthase genes can therefore the sequence homology using the on the plasmid pin 5-49 contained cDNA or its parts or the Se Sequence information according to SEO ID No: 1 in the usual way with the known methods of molecular biology Plants can be isolated in a simple manner.

As plants from which Pinosylvin according to the invention synthase genes that come in handy come in handy all monocotyledonous or dicotyledonous plants, preferably dicotyledonous plants in question, being exemplary and preferably pine (Pinus sp.) and particularly preferred Pinus sylvestris may be mentioned.

As already mentioned, Pinosylvin synthase genes or their coding region preferred, that hybridize to the cDNA that is on the plasmid pin 5-49. The gene or coding region of the The gene can be obtained with the help of the cDNA in the usual way will.  

The Escherichia coli strain E. coli pin 5-49 contains this Plasmid pin 5-49. This strain was with the German Collection of microorganisms (DSM), Mascheroder Weg 1b, D-3300 Braunschweig, Federal Republic of Germany in About in accordance with the provisions of the Budapest Treaty international recognition of the deposit of microorganisms for the purposes of patent procedures deposited (filing date: August 29, 1991). He he held the accession number DSM 6689.

This strain and its mutants are also part of it of the present invention. That behind in this host put plasmid pin 5-49 in the usual way through the Propagation of the trunk and subsequent isolation of the Plasmids easily obtained in the required amounts will.

Functionally complete genes, such as those according to the invention Pinosylvin synthase genes consist of a regulato risch acting part (in particular promoter) and the Structural gene, which is the protein pinosylvine synthase encoded.

Both gene parts can be used independently will. So it is possible the regulatory part one (different from the pinosylvine synthase gene) another Subsequent DNA sequence, which after installation in the plant genome is to be expressed. Because only relative few isolated promoters are known which their Can have an effect in plants or plant cells the promoters of the pinosylvine synthase genes, which are also components of the present invention  are valuable tools in generating transfor plants or plant cells.

It is also possible to structure the pinosylvine synthase Genes a "foreign" regulatory part switch on. This could be advantageous if at be did plants only certain (e.g. plant-specific) regulatory genes become sufficiently effective can. The pinosylvin synthase structure genes represent thus represent valuable, independently usable units and, as already stated, are also part of the previous lying invention. The Pinosylvin according to the invention synthase genes can be inserted into the regulatory parts and the structural genes ge be separated. It is also possible to different parts of the naturally occurring pinosylvine synthase genes to new functional "synthetic" genes kidneys. The completely natural ones are preferred Pinosylvine synthase genes according to the invention (or the gene Units).

With the help of the usual methods it is possible to Pinosylvine synthase genes (or the gene units) or their parts one or more times (e.g. tandem arrangement), preferably simple, in any prokaryotic (preferably bacterial) or eukaryotic (pre preferably vegetable) DNA as "foreign" or "additional "DNA". For example, the protein code regulatory DNA are provided and built into plants. The "modified" recombinant DNA, which e.g. B. to transform Plants or plant cells can be used and  after transformation in plants or plant cells is part of the present inven dung.

The Pinosylvinsynthase genes (or the gene units) and / or their parts as well as the recombinant DNA as "foreign" or "additional" DNA in vectors (esp special plasmids, cosmids or phages), in transfor mated microorganisms (preferably bacteria, ins special Gram-negative bacteria, such as E. coli) as well in transformed plant cells and plants or in whose DNA is included. Such vectors, transform microorganisms (which also contain these vectors can) as well as the transformed plant cells and Plants and their DNA are components of this ing invention.

As already indicated, according to the invention the pino sylvinsynthase genes (or the gene units) one or multiple times (in the same or different places of the Genome) built into the natural plant genome, whereby different genes are also combined with each other can be. For plants that already have the Ability of the Pinosylvinsynthase synthesis can feature the installation of one or more Pino according to the invention sylvine synthase genes at a significantly improved level Lead resistance behavior. For plants that do not have a pino Sylvin synthase genes are included by incorporation such genes also have increased pest resistance reached. If necessary, only those according to the invention Structural genes are used, one possibly from the respective  lig plant isolated regulatory DNA element is connected upstream.

The increased resistance of the transform according to the invention Plant cells and plants is important for Agriculture and forestry, for growing ornamental plants, medicinal plant cultivation and plant breeding. Also at the cultivation of plant cells, e.g. B. for extraction of pharmaceutically usable substances, it is from before partly to have plant cells available which are against infestation by microbial pests, in particular Fungi that show increased resistance.

The present invention thus also relates to a method for producing transformed plant cells (including protoplasts) and plants (including plant parts and seeds) with increased resistance to pests, which is characterized in that
(a) one or more pinosylvine synthase genes (or gene units) and / or parts of the pinosylvine synthase genes (or gene units) and / or recombinant DNA according to the invention is used in the genome of plant cells (including protoplasts) and if necessary
(b) from the transformed plant cells (including protoplasts), completely transformed plants are regenerated and, if appropriate, propagated and if appropriate
(c) the desired plant parts (including seeds) are obtained from the transformed plants of the parent generation or further generations obtained therefrom.

The process steps (a), (b) and (c) can be after knew procedures and methods in the usual way be performed.

Transformed plant cells (including Protoplas ten) and plants (including parts of plants and Sa men), which contain one or more pinosylvine synthase genes (or gene units) and / or parts of Pinosylvinsyn thase genes (or the gene units) as "foreign" or Contain "additional" DNA as well as such transformed Plant cells and plants, which according to the above ver driving are also included the invention.

Parts of the present invention are also:
(a) Use of the pinosylvine synthase genes (or the gene units) and / or their parts and / or the recombinant DNA according to the invention and / or the recombinant vectors according to the invention and / or the transformed microorganisms according to the invention for the transformation of plant cells (including Protoplasts) and plants (including parts of plants and seeds) which
(b) Use of the transformed plant cells (including protoplasts) and plants (including plant parts and seeds) according to the invention for the production of propagation material and for the production of new plants and their propagation material which
(c) Use of the Pinosylvinsyn thase genes according to the invention (or the gene units) and / or their parts and / or the recombinant DNA according to the invention for combating pests and the
d) Use of the cDNA or its parts contained on the plasmid pin 5-49 and of the DNA sequences corresponding to the sequence information according to the sequence listing SEO ID NO: 1 for the isolation of Pinosyl vinsynthase genes or their parts from plants and for the determination of Pinosylvinsynthase- Genes in plants and (in general) in the production of transformed plant cells (including protoplasts) and plants (including plant parts and seeds).

A number of different methods are available the Pinosylvinsynthase genes or the gene units or their parts as "foreign" or "additional" DNA in the genetic material of plants or plant cells to use. The gene transfer can be done according to the general practice Lichen known methods take place, the expert the appropriate method without difficulty can average.

The Ti plasmid from Agrobacterium tumefaciens stands as particularly inexpensive and widely applicable vector for  Transfer of foreign DNA into genomes dicotyledonous and mo nokotyler plants available. The genetic mate rial, which codes for pinosylvine synthase, becomes together with regulatory DNA sequences in the T-DNA of suitable Ti plasmids (e.g. Zambryski et al. 1983) and by infection of the plant, infection of parts of plants or plant tissues, such as. B. from Leaf disks, stems, hypocotyls, cotyledons, Meri stemen and derived tissues such. B. secondary ren embryos and calli or by co-culture of proto plastic with Agrobacterium tumefaciens.

An alternative is to incubate purified DNA, which contains the desired gene in plant protoplasts (e.g. Hain et al., 1985; Krens et al., 1982; Paszkowski et al., 1984) in the presence of polycations or Calzi salt and polyethylene glycol.

The DNA uptake can also by an electri field (electroporation) are favored (e.g. Fromm et al., 1986).

The DNA can also be obtained from plant pollen in a known manner be introduced by physically pollen with be small particles are "shot at", which the Carry DNA (see EP-A 02 70 356).

The plants are regenerated in a known manner with the help of suitable nutrient media (e.g. Nagy and Maliga 1976).  

In a preferred embodiment of the fiction method (according to the method from EP-A 1 16 718) are the genes or gene units that are complementary to cDNA from pin 5-49 are present in the genus of Pinus, in isolated form in a suitable intermediate E. coli vector e.g. B. pGV700 or pGV710, (see EP-A- 1 16 718 or preferably derivatives thereof, the additional Lich a reporter gene such as B. nptII (Herrera-Estrella et al. 1983) or hpt (Van den Elzen et al 1986) ent hold, cloned.

The plasmid thus constructed is based on Agrobacterium tume faciens, the z. B. pGV 3850 or derivatives thereof (Zambryski et al. 1983) using conventional methods (e.g. Van Haute et al. 1983). Alternatively, the Pinosylvine synthase gene unit in a binary vector, e.g. B. pCV001 or pCV002 (e.g. Koncz and Schell 1986) cloned and into a suitable one as described above Agrobacterium strain (Koncz and Schell 1986) transferred will. The resulting Agrobacterium strain, which is the Pinosylvine synthase genes or gene units in one Plants contains transferable form in the following used for plant transformation.

In a further preferred embodiment, the given isolated Pinosylvinsynthase gene units if together with another plasmid that contains a report tergen for plant cells, e.g. B. for kanamycin resistance (e.g. Herrera-Estrella et al. 1983) or a hygromy cin resistance (van den Elzen, 1986), preferably see pLGV neo 2103 (Hain et al. 1985), pMON 129 (Fraley  R.T. et al., Proc. National Acad. Sci. USA 80, 4803 (1983), pAK 1003, pAK 2004 (Velten J. et al., EMBO Journ. Vol. 3, 2723 (1984) or pGSST neo 3 (pGSST3) (EP-A-1 89 707), in the usual way by direct gene transfer to plant protoplasts (e.g. grove et al 1985). The plasmid or plasmids in zir more culinary, but preferably in a linear form gen. When using a plasmid with reporter gene then kanamycin-resistant protoplasts on expresses sion of Pinosylvinsynthase checked. Otherwise (without reporter gene) the resulting calli will show up the expression of the Pinosylvinsynthase genes checks (screening with usual methods).

Transformed (transgenic) plants or plant cells are according to the known methods, for. B. by leaf slice transformation (e.g. Horsch et al. 1985) Co-culture of regenerating plant protoplasts or cells cultures with Agrobacterium tumefaciens (e.g. Marton et al. 1979, Hain et al. 1985) or by direct DNA Transfection generated. Resulting transformed Plants are selected either by selection on the express sion of the reporter gene, e.g. B. by phosphorylation of kanamycin sulfate in vitro (Reiss et al. 1984; Schreier et al. 1985) or through the expression of the No palinsynthase (after Aerts et al. 1983) or Pinosylvin synthase by Northern blot analysis and Western blot Analysis proven. The Pinosylvinsynthase and the Stilbenes can also be saved in a known manner specific antibody in transformed plants be directed. Pinosylvine synthase can also by Enzyme activity test can be detected (Gehlert et al.,  The cultivation of the transformed plant cells like this how the regeneration to complete plants takes place according to the generally accepted methods with the help of because suitable nutrient media.

Both the transformed plant cells and the transformed plants, which the invention Pinosylvinsynthase genes (or the gene units) ent and what components of the present inven are significantly more resistant to Pests, in particular phytopathogenic fungi.

In the context of the present invention means the expression "plants" both whole plants as well also parts of plants, such as leaves, seeds, bulbs, stick linge etc. "plant cells" include protoplasts, Cell lines, plant calli, etc. "Propagation mat rial "means plants and plant cells which are used for Propagation of the transformed plants and plants cells can be used and is therefore also Part of the present invention.

In the present context, the expression "in essential equivalent DNA sequences "that the Er invention also includes such modifications in which the function of the Pinosylvinsynthase genes and their parts is not so affected that pinosylvine synthase is no longer formed or the regulatory gene part no longer takes effect. Corresponding modifications can be replaced, added and / or replaced  Removal of DNA fragments, individual codons and / or individual nucleic acids.

In the microorganisms that can be used according to the invention "mutants" indicates such modified microorganisms, which is still essential for the implementation of the invention Liche features, especially the plasmid pin 5-49 included.  

Regarding the plants, which by the installation (Transforma tion) of the Pinosylvinsynthase genes according to the invention (or the gene units) resistance or an increased Resistance to the pests are conferred can include practically all plants. A special Naturally, there is a need to create resistance the crops, such as forest plants, e.g. B. spruce, Fir, Douglas fir, pine, larch, beech and oak as well as plants and plants that supply food and raw materials, e.g. B. cereals (in particular wheat, rye, barley, ha fer, millet, rice and corn), potato, legumes (such as Legumes and in particular alfalfa, soybeans), Ge vegetables (especially cabbages and tomatoes), fruit (esp special apples, pears, cherries, grapes, citrus, Pineapple and banana), oil palms, tea, cocoa and coffee shrubs, tobacco, sisal and cotton as well as healing plants like Rauwolfia and Digitalis. Especially before Potatoes, tomatoes and legumes are mentioned. The Pinosylvin according to the invention are preferred synthase genes as "foreign" DNA in the genome of plants built-in.

As pests, against which with the help of the Invention moderate Pinosylvinsynthase genes resistance, or increased Resistance can be achieved are animal Pests, such as insects, mites and nematodes, and mi crobial pests, such as phytopathogenic fungi, bacteria called viruses and viruses. Be particularly highlighted microbial pests, especially phytopathogenic Mushrooms.  

The harmful insects include in particular Insects of the orders:

Orthoptera, Dermaptera, Isoptera, Thysanoptera, Heteroptera, homoptera, lepidoptera, coleoptera, Hymenoptera and Diptera.

The harmful mites include in particular: Tarsonemus spp., Panonychus spp. and Tetranychus spp.

The harmful nematodes include in particular: Pratylenchus spp., Heterodera spp. and Meloidogyne spp.

The microbial pests include in particular the phytopathogenic mushrooms:

Plasmodiophoromycetes, Oomycetes, Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes, Deuteromy cetes.

The phytopathogenic bacteria include in particular the Pseudomonadaceae, Rhizobiaceae, Enterobacteriaceae, Corynebacteriaceae and Streptomycetaceae.

Virus diseases include in particular mosaic, Dwarfism and yellowing viruses.

Some pathogens are exemplary but not limiting of viral, fungal and bacterial diseases, which fall under the generic terms listed above, called:  

Barley Yellow Dwarf Virus (BYDV), Potato Virus Y (PVY), Cucumber Mosaic Virus (CMV), Watermelon Mosaic Virus (WMV), Tristeza virus, Tobacco Mosaic Virus (TMV), Tobacco Necrosis Virus (TNV), bed of necrotic yellow vein Virus (BNYVV), Rhizomania virus.

Xanthomonas species, such as, for example, Xanthomonas campestris pv. Oryzae;
Pseudomonas species, such as, for example, Pseudomonas syringae pv. Lachrymans;
Erwinia species, such as, for example, Erwinia amylovora; Pythium species, such as, for example, Pythium ultimum; Phytophthora species, such as, for example, Phytophthora infestans;
Pseudoperonospora species, such as, for example, Pseudoperonospora humuli or Pseudoperonospora cubense;
Plasmopara species, such as, for example, Plasmopara viticola;
Peronospora species, such as, for example, Peronospora pisi or P. brassicae;
Erysiphe species, such as, for example, Erysiphe graminis; Sphaerotheca species, such as, for example, Sphaerotheca fuliginea:
Podosphaera species, such as, for example, Podosphaera leuco tricha;
Venturia species, such as, for example, Venturia inaequalis; Pyrenophora species, such as, for example, Pyrenophora teres or P. graminea
(Conidial form: Drechslera, Syn: Helminthosporium);
Cochliobolus species, such as, for example, Cochliobolus sativus
(Conidial form: Drechslera, Syn: Helminthosporium);
Uromyces species, such as, for example, Uromyces appendiculatus;
Puccinia species, such as, for example, Puccinia recondita;
Tilletia species, such as, for example, Tilletia caries;
Ustilago species, such as, for example, Ustilago nuda or Ustilago avenae;
Pellicularia species, such as, for example, Pellicularia sasakii;
Pyricularia species, such as, for example, Pyricularia oryzae;
Fusarium species, such as, for example, Fusarium culmorum;
Botrytis species, such as, for example, Botrytis cinerea;
Septoria species, such as, for example, Septoria nodorum;
Leptosphaeria species, such as, for example, Leptosphaeria nodorum;
Cercospora species, such as, for example, Cercospora canescens;
Alternaria species, such as, for example, Alternaria brassicae;
Pseudocercosporella species, such as, for example, Pseudocerco sporella herpotrichoides. Helminthosporium carbonum is also listed.

The present invention is based on the following exemplary embodiments are explained in more detail.

1. Isolation of the Pinosylvinsynthase Gene from Pinus

Plants and cell cultures from Pinus (Pinus sylvestris) contain the genes for Pinosylvinsynthase, which the Formation of pinosylvine synthase (size of the protein 4500 D; Reaction with specific antiserum) (Gehlert et al. 1990).

When isolating the Pinosylvinsynthase genes the known methods and methods of molecular bio logic used, such as in the following Manual described in detail; Sambrook, J., Fritsch, E.F., Maniatis, T .: Molecular Cloning: A Labo ratory manual; Cold Spring Harbor Laboratory, Second Edition 1989.

First there will be a "gene library" for Pinus placed: Genomic DNA from enriched cell nuclei (Bedbrook, J., Plant Molecular Biology Newsletter 2, 24, 1981) is so ge with the restriction enzyme NdeII cut that DNA fragments with an average lengths of about 12,000 nucleotide pairs arise. These Fragments are placed in the BamHI site of Lambda phage EMBL4 cloned (Frischauf et al., J. Mol. Biol. 170, 827-842, 1983), and the phages are ver in E. coli increases. The entirety of the phage population contains cloned in sections,  the entire genomic DNA of the pinus cells, and thus also the genes for pinosylvine synthases (Multigen family).

The genes for Pinosylvinsynthase, their mRNA and the Pinosylvine synthase cDNA each contain the same Nucleic acid sequences as they are derived from each other can be (gene → mRNA → cDNA). This means that the Genes for Pinosylvinsynthase by Specific Hybridi sation with the respective pinosylvine synthase cDNA or can be identified with specific oligonucleotides. The phages with the genes are hybridized identified, then isolated and multiplied. The one in this Phage cloned genomic DNA from Pinus continues by analysis with various restriction enzymes kar tiert, and the position of the Pinosylvinsynthase genes through further hybridization experiments with cDNA Festge sequences or synthetic oligonucleotides sets. Eventually, the gene units are digested cut out of the phage with restriction enzymes ten, in the correspondingly cut plasmid vector pUC18 cloned (Gibco-BRL GmbH, Eggenstein, Federal Republic Germany), and increased as recombinant plasmids.

2. Description of the plasmids pin 5-49 (see FIG. 1

The plasmid consists of two components:  

• (i) cDNA (partial sequence) of pinosylvine synthase: the cDNA inserted into the plasmid pT7 / T3 is 1.3 kb long and can be used with EcoRI from the Plasmid pin 5-49 are cut out.
• (ii) Vector plasmid: The cDNA is in the vector pT7 / T3 (Pharmacia LKB GmbH, Freiburg Federal Republic Germany) cloned. The size of the vector is 2800 nucleotide pairs. He carries the gene for ampi cillin resistance, d. H. E. coli cells with this Plasmid grow in nutrient media containing the antibiotic Ampicillin included. Ori: term for sequences zen for the propagation of the plasmid in E. coli are necessary.

The plasmid pin 5-49 carries a gene for ampicillin resi stenz and contains the above as Pinosylvinsynthase cDNA described EcoRI fragment with about 1.3 kb. It can be in E. coli cells containing pin 5-49 (E. coli pin 5-49), be propagated in the usual way.

Preferred nutrient medium for E. coli cells (e.g. JA221, Nakamura, K., Inouye, M., EMBO J. 1, 771-775, 1982) which contain pin 5-49 (E. coli pin 5-49):

3. Tobacco transformation a) Culture of tobacco sprouts and isolation of tobacco protoplasts

Nicotiana tabacum (Petit Havana SR1) is considered sterile shoot culture on hormone-free LS medium (Linsmaier and Skoog 1965) increased. At intervals From about 6-8 weeks, shoot sections are opened fresh LS medium implemented. The shoot cultures with 12 h light (1000-3000 lux) in one Culture room kept at 24-26 ° C.

For the isolation of leaf protoplasts, approx. 2 g of leaves (approx. 3-5 cm long) are cut into small pieces (0.5 cm × 1 cm) with a fresh razor blade. The leaf material is in 20 ml enzyme solution, consisting of K3 medium (Nagy and Maliga 1976), 0.4 m sucrose, pH 5.6, 2% cellulase R10 (Serva), 0.5% Macerozym R10 (Serva) for 14 Incubated for 16 h at room temperature. The protoplasts are then separated from cell residues by filtration through 0.30 mm and 0.1 mm steel sieves. The filtrate is centrifuged at 100 × g for 10 minutes. During this centrifugation, intact protoplasts float and collect in a band at the top of the enzyme solution. The pellet from cell residues and the enzyme solution are aspirated with a glass capillary. The pre-cleaned protoplasts are made up to 10 ml with fresh K3 medium (0.4 M sucrose as an osmotic agent) and floated again. The washing medium is suctioned off and the protoplasts are diluted to 1-2 × 10 ⁵ / ml for culture or subsequent infection with agrobacteria (coculture). The protoplast concentration is determined in a counting chamber.

b) Transformation of regenerating tobacco prototype plastify by co-culture with Agrobacterium tumefaciens:

The method of Marton et al. 1979 used with minor changes. The protoplasts are isolated as described and in a density of 1-2 × 10 ⁵ ml in K3 medium (0.4 m sucrose, 0.1 mg / l NAA, 0.2 ml in K3 medium (0.4 m Sucrose, 0.1 mg / l NAA, 0.2 mg kinetin) incubated for 2 days in the dark and for one to two days under low light (500 lux) at 26 ° C. As soon as the first divisions of the protoplasts occur, 30 µl of a Agrobacterium suspension in mini A (Am) medium (density approx. 10 ⁹ agrobacteria / ml) added to 3 ml of regenerating protoplasts.

The coculture time is 3-4 days at 20 ° C in the dark. The tobacco cells are then filled into 12 ml centrifuge tubes, diluted to 10 ml with sea water (600 mOsm / kg) and pelleted at 60 × g for 10 minutes. This washing process is repeated 1-2 more times to remove most of the agrobacteria. The cell suspension is in a density of 5 × 10 ⁴ / ml in K3 medium (0.3 m sucrose) with 1 mg / l NAA (naphthyl-1-acetic acid), 0.2 mg / l kinetin and 500 mg / l des Cephalosporin antibiotic cefotaxime cultivated. The Zellsus pension is diluted every week with fresh K3 medium and the osmotic value of the medium is gradually reduced by 0.05 m sucrose (approx. 60 mOsm / kg) per week. Selection with kanamycin (100 mg / l kanamycin sulfate (Sigma), 660 mg / g active km) is started 2-3 weeks after coculture in agarose "bead type culture" (Shillito et al. 1983). Colonies resistant to kanamycin can be distinguished from the background of remaining colonies 3-4 weeks after the start of the selection.

c) Direct transformation of tobacco protoplasts with DNA. Calcium nitrate-PEG transformation.

Approx. 10 ⁶ protoplasts in 180 μ 1 K3 medium with 20 μl aqueous DNA solution containing 0.5 μg / μl plasmid which carries the genomic pinosylvine synthase gene and 0.5 μg / μl pLGV neo 2103 (Hain et al. 1985) contains, carefully mixed. Subsequently, 200 ul fusion solution (0.1 m calcium nitrate, 0.45 M mannitol, 25% polyethylene glycol (PEG 6000), pH 9) are carefully added. After 15 minutes, 5 ml of washing solution (0.275 M calcium nitrate pH 6) are added and after a further 5 minutes the protoplasts are transferred into a centrifuge tube and pelleted at 60 × g. The pellet is taken up in a small amount of K3 medium and cultured as described in the next section. Alternatively, the protoplasts according to Hain et al. 1985 transformed who.

The transformation can also be done without the addition of the 0.5 µg / µl pLGV neo 2103 can be performed. There in in this case no reporter gene is used, the resulting calli on the Presence of the pinosylvine synthase gene unit checked using a dot blot hybridization. As a hybridization sample, the cDNA sequence is over pin 5-49 can be used. Of course you can too other detection methods, such as testing with antibodies or finding fungal resistance will.

d) culture of the protoplasts incubated with DNA and Selection of kanamycin resistant calli

For the culture and selek described below colony resistant kanamycin becomes a modi "Bead Type culture" technique (Shillito et al. 1983) used. A week after treatment of the Protoplasts with DNA (see c) become 3 ml of the cell suspension with 3 ml K3 medium (0.3 M sucrose Hormones; 1.2% (Seaplaque) LMT agarose (low melting agarose, marine colloids) in 5 cm petri dishes mixes. For this purpose, agarose becomes dry auto piano and after adding K3 medium in the micro  wave cooker briefly boiled. After the The agarose slices ("beads") are used with agarose the embedded tobacco micro calli for more Culture and selection in 10 cm petri dishes trans and 10 ml K3 medium (0.3 M sucrose, 1 mg / l NAA, 0.2 mg / l kinetin) and 100 mg / l kana mycin sulfate (Sigma) added. The liquid medium will changed every week. The osmotic Value of the medium gradually reduced.

The exchange medium (K3 + Km) is changed every week 0.05 m of sucrose (approx. 60 mOsm) reduced.

Scheme of selection of kanamycin resistant tobacco colonies after DNA transformation:

e) Regeneration of kamaycin-resistant plants

Once the kanamycin-resistant colonies become one Have reached a diameter of approx. 0.5 cm Half on regeneration medium (LS medium, 2% Sucrose, 0.5 mg / l benzylaminopurine BAP) and with 12 h light (3000-5000 lux) and 24 ° C in Cultural space kept. The other half is called Callus culture on LS medium with 1 mg / l NAA, 0.2 mg / l Kinetin, 0.1 mg / l BAP and 100 mg / l kanamydine sulfate propagates. When the regenerated shoots about 1 cm are large, they are cut off and put on 1/2 LS Medium (1% sucrose, 0.8% agar) without growth regulators set for rooting. The sprout are on 1/2 MS medium with 100 mg / l kanamycin rooted sulfate and later converted into soil.

f) transformation of leaf disks by agro bacterium tumefaciens

For the transformation of leaf discs (Horsch et al. 1985) leaves of approx. 2-3 cm long are cut from sterile shoot cultures into discs of 1 cm in diameter and with a suspension of appropriate Agrobacteria (approx. 10 ⁹ / ml) (cf. b) incubated in Am medium, see below) for about 5 minutes. The infected leaf pieces are kept on MS-Mediam (see below) without hormones for 3-4 days at approx. 24 ° C. During this time, Agrobacterium grows over the leaf pieces. The leaf pieces are then washed in MS medium (0.5 mg / ml BAP, 0.1 mg / ml NAA) and on the same medium (0.8% agar) with 500 μg / ml cefotaxime and 100 μg / ml kanamycin sulfate (Sigma) placed. The medium should be replaced after two weeks. Transformed shoots become visible after another 2-3 weeks. The regeneration of sprouts should also be carried out in parallel without selection pressure. The regenerated rung must then be tested by biological tests such. B. on nopaline synthase or Pinosylvin synthase activity on transformation can be tested. In this way 1-10% transformed shoots are obtained.

Biochemical detection method of transformation Detection of nopaline in plant tissues

As with Otten and Schilperoort (1978) and Aerts et al. (1979) as follows. 50 mg of plant material (callus or leaf pieces) are incubated overnight in LS medium with 0.1 M arginine at room temperature in an Eppendorf vessel. The plant material is then dabbed onto absorbent paper, homo-genized in a fresh Eppendorf centrifuge tube with a glass rod and centrifuged for 2 minutes in an Eppendorf centrifuge. 2 .mu.l of the supernatant are applied in spots on a paper suitable for electrophoresis (Whatman 3 MM paper) (20 × 40 cm) and dried. The paper is soaked with the eluent (5% formic acid, 15% acetic acid, 80% H ₂ O, pH 1.8) and electrophoresed at 400 V for 45 minutes. Nopalin runs towards the cathode. The paper is then hot dried with a stream of air and drawn in the direction of travel through phenanthrenequinone colorant (equal volume 0.02% phenanthrenequinone in ethanol and 10% NaOH in 60% ethanol). The dried paper is viewed and photographed under long-wave UV light. Arginine and arginine derivatives are stained yellow fluorescent with the reagent.

Neomycin phosphotransferase (NPT II) enzyme test

NPT II activity in plant tissue is determined by in situ phosphorylation of kanamycin, as described by Reiss et al. (1984) and by Schreier et al. (1985) modified as follows. 50 mg of plant tissue are homogenized in 50 .mu.l extraction buffer (10% glycerol, 5% 2-mercaptoethanol, 0.1% SDS, 0.025% bromophenol blue, 62.5 mM Tris pH 6.8) with the addition of glass powder on ice and for 10 minutes centrifuged in an Eppendorf centrifuge at 4 ° C. 50 ul of the supernatant are placed on a native polyacrylamide gel (145 × 110 × 1.2 mm; separating gel: 10% acrylamide, 0.33% bisacrylamide, 0.375 M Tris pH 8.8, stacking gel: 5% acrylamide, 0.165% bisacrylamide, 0.125 M Tris pH 6.8) applied and electrophoresed overnight at 4 ° C and 60 V. As soon as the bromophenol blue marker runs out of the gel, the gel is washed twice with distilled water for 10 minutes and once for 30 minutes with reaction buffer (67 mM tris-maleate, pH 7.1, 42 mM MgCl ₂ , 400 mM ammonium chloride ). The gel is placed on a glass plate of the same size and covered with 40 ml of 1% agarose in reaction buffer which contains the substrates kanamycin sulfate (20 µg / ml) and 20-200 µCi ³² P ATP (Amersham). The sandwich gel is incubated for 30 minutes at room temperature and then a sheet of phosphocellulose paper P81 (Whatman) is placed on the agarose. Four filter papers of 3 MM, (Whatman) and some paper towels are stacked on top. The transfer of in situ phosphorylated radioactive kanamycin phosphate to the P81 paper is stopped after 3-4 h. The P81 paper is for 30 min. incubated in a solution of Proteinase K and 1% sodium dodecyl sulfate (SDS) at 60 ° C and then washed 3-4 times in 250 ml of 10 mM phosphate buffer pH 7.5 at 80 ° C, dried and for 1-12 h autoradiographed at -70 ° C (XAR5 film Kodak).

4. Transformation of Solanum tuberosum (potato)

The transformation was carried out exactly according to that in the EP A-02 42 246, pages 14 to 15 indicated manner transformed, the agrobacteria Ti plasmids contain the Pinosylvinsynthase genes carry.

Obtain all percentages in the examples above per cent by weight unless otherwise stated becomes.  

In the plants obtained according to the above examples cells and plants (tobacco) was the presence of the Pinosylvin synthase genes by Southern blot analysis approved. Expression of the Pinosylvinsynthase Genes was by Northern blot analysis, Pinosylvinsynthase and Pinosylvin with the help of specific antibodies proven. Transformed and non-transformed Plants (for comparison) were scored with a spore pension sprayed by botrytis cinera and after 1 week the fungal infection is rated. The transformed plants showed (compared to the non-transformed comparison plants) an increased resistance to fungal attack.

The following are some of those in the transformation Media used by plants or plant cells described:

Am medium

3.5 g K₂HPO₄
1.5 g KH₂PO₄
0.5 g Na₃ citrate
0.1 g MgSO₄ × 7 H₂O
1 g (NH₄) ₂SO₄
2 g glucose
ad 1 l

Medium for sterile shoot culture of tobacco

Macro elements ½ of the concentration of MS salts
Micro elements ½ of the concentration of MS salts
Fe-EDTA Murashige and Skoog (MS)
Myo-inositol 100 mg / l
Sucrose 10 mg / l
Agar 8 g / l

Ca panthotenate 1 mg / l
Biotin 10 mg / l
Nicotinic acid 1 mg / l
Pyridoxine 1 mg / l
Thiamine 1 mg / l
pH 5.7 before autoclaving

K3 medium

On the culture of Nicotiana tabacum petit Havana SR1, Nicotiana tabacum Wisconsin 38, and Nicotiana plumbaginifolia protoplasts (Nagy and Maliga, 1976)

Macro elements
NH₄NO₃ 250 mg / l
KNO₃ 2500 mg / l
CaCl₂ · 2 H₂O 900 mg / l
MgSO₄ · 7 H₂O 250 mg / l
NaH₂PO₄ · 1 H₂O 150 mg / l
(NH₄) ₂SO₄ 134 mg / l
CaHPO₄ · 1 H₂O 50 mg / l

Micro elements
H₃BO₃ 3 mg / l
MnSO₄ · 1 H₂O 10 mg / l
ZnSO₄ · 4 H₂O 2 mg / l
KI 0.75 mg / l
Na₂MoO₄ · 2 H₂O 0.25 mg / l
CuSO₄ · 5 H₂O 0.025 mg / l
CoCl₂ · 6 H₂O 0.025 mg / l

Fe-EDTA
Na₂EDTA 37.2 mg / l
FeSO₄ · 7 H₂O 27.8 mg / l

Inositol 100 mg / l
Sucrose 137 g / l (= 0.4 M)
Xylose 250 mg / l

Vitamins
Nicotinic acid 1 mg / l
Pyridoxine 1 mg / l
Thiamine 10 mg / l

Hormones
NAA 1.0 mg / l
Kinetin 0.2 mg / l
pH 5.6
Sterilize the filter

Linsmaier and Skoog Medium (Linsmaier and Skoog 1965)

For the culture of regenerating protoplasts and for Ge weaving culture of tobacco tumors and callus. Linsemaier and Skoog (LS) Medium is Murashige and Skoog Medium (Murashige and Skoog, 1962) with the following modifi cations:

• - Thiamine is weighed in a higher concentration 0.4 mg / l instead of 0.1 mg / l;
• - Glycine, pyridoxine and nicotinic acid are missing.

Macro elements
NH₄NO₃ 1650 mg / l
KNO₃ 1900 mg / l
CaCl₂ · 2 H₂O 440 mg / l
MgSO₄ · 7 H₂O 370 mg / l
KH₂PO₄ 170 mg / l

Micro elements
H₃BO₃ 6.2 mg / l
MnSO₄ · 1 H₂O 22.3 mg / l
ZnSO₄ · 4 H₂O 8.6 mg / l
KI 0.83 mg / l
Na₂MoO₄ · 2 H₂O 0.25 mg / l
CuSO₄ · 5 H₂O 0.025 mg / l
CoCl₂ · 6 H₂O 0.025 mg / l

Fe-EDTA
Na₂EDTA 37.2 mg / l
FeSO₄ · 7 H₂O 27.8 mg / l

Inositol 100 mg / l
Sucrose 30 mg / l
Agar 8 g / l
Vitamins
Thiamine 0.4 mg / l

Hormones:
NAA 1 mg / l
Kinetin 0.2 mg / l
pH 5.7 before autoclaving

The following literature can be cited for the transformation of plants or plant cells:
Aerts M, Jacobs M, Hernalsteens JP, Van Montagu M, Schell J (1983) Induction and in vitro culture of Arabidopsis thaliana crown gall tumors. Plant Sci Lett. 17: 43-50

Fromm ME, Taylor LP, Walbot V (1986) Stable transfor mation of maize after gene transfer by electroporation. Nature 319: 791-793  

Gehlert, R., Schöppner, A., and Kindl, H. (1990) Synthase from Seedlings of Pinus sylvestris: Purification and Induction in Response to Fungal Infection. Molecular Plant-Microbe Interactions. Vol 3, No 6, p.p. 444-449

Hain, R., Stabel, P., Czernilofsky, A. Pp., Steinbiß, H.H., Herrera-Estrella, L., Schell, J. (1985) Uptake, integration, expression and genetic transmission of a selectable chimeric gene by plant protoplasts. Molec gene Genet 199; 161-168

Herrera-Estrella L., De Block M., Messens E., Hernal steens JP., van Montagu M., Schell J. (1983) EMBO J. 2: 987-995.

Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 277: 1229-1231 Krens FH, Molendÿk L, Wullems GJ, Schilperoort RA (1982) in vitro transformation of plant protoplasts with Ti plasmid DNA. Nature 296: 72-74

Koncz C, Schell J (1986) The promotor of T _L -DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a noval type of Agrobacterium binary vector. Mol. Gen. Genet. (1986) 204: 338-396

Linsmaier DM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18: 100-127

Marton L, Wullems GJ, Molendÿk L, Schilperoort PR (1979) In vitro transformation of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens. Nature 277: 1229-131

Murashige, T. and Skoog F. (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plans. 15, 47

Nagy JI, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z Plant Physiol 78: 453-455

Otten LABM, Schilperoort RA (1978) A rapid microscale method for the detection of Lysopin and Nopalin dehydrogenase activities. Biochim biophys acta 527; 497-500 Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I (1984) Direct gene transfer to plants. EMBO J 3: 2717-2722

Shillito RD, Paszkowski J. Potrykus I (1983) Agarose plating and Bead type culture technique enable and stimulate development of protoplast-derived colonies in an number of plant species. Pl Cell Rep 2: 244-247  

Van den Elzen PJM, Townsend J, Lee KY, Bedbrook JR (1985) Achimaeric resistance gen as a selectable marker in plant cells. Plant Mol. Biol. 5, 299-302.

Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) Intergenic transfer and exchange recom bin of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of Ti plasmids of / Agrobacterium tumefaciens. EMBO J 2: 411-418

Velten J, Velten L, Hain R, Schell J (1984) Isolation of a dual plant promotor fragment from the Ti Plasmid of Agrobacterium tumefaciens. EMBO J 12; 2723-2730

Wullems GJ, Molendÿk L, Ooms G, Schilperoort RA (1981) Differential expression of crown gall tumor markers in transformants obtained after in vitro Agrobacterium tumefaciens - induced transformation of cell wall regenerating protoplasts derived from Nicotiana tabacum. Proc Natl Acad Sci 78; 4344-4348

Zambryski P, Joos H, Genetello C, van Montagu M, Schell J (1983) Ti-plasmid vector for the introduction of DNA into plant cells without altering their normal regeneration capacity, EMBO J 12; 2143-2150.

Reiss B, Sprengel R, Will H and Schaller H (1984) A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell tracts, GENE 1081: 211-217  

Schreier P, Seftor E, Schell J and Bohnert H (1985) The use of nuclear-encoded sequences to direct the light regulated synthesis and transport of a foreingn protein into plant chloroplasts, EMBO J Vol. 4, No. 1: 25-32

Furthermore, the following published patents registrations are listed:

EP-A 1 16 718
EP-A 1 59 418
EP-A 1 20 515
EP-A 1 20 516
EP-A 1 72 112
EP-A 1 40 556
EP-A 1 74 166
EP-A 1 22 791
EP-A 1 26 546
EP-A 1 64 597
EP-A 1 75 966
WO 84/02 913
WO 84/02 919
WO 84/02 920
WO 83/01 176

Explanations to FIG. 1

Fig. 1 represents the plasmid pin 5-49. The Pinosyl vinsynthase cDNA is located on the approximately 1.3 kb EcoRI fragment.

In Fig. 1 where:
E: EcoRI
B: Bam HI
H: Hind III
Pl: Polylinker from the plasmid pT7 / T3

SEQ ID NO: 1
TYPE OF SEQUENCE: nucleotide with corresponding peptide
SEQUENCE LENGTH: 570 base pairs
STRANGEFORM: single strand
TOPOLOGY: linear
MOLECULE TYPE: cDNA
ORIGINAL ORIGIN:
ORGANISM: Pinus sylvestris
DIRECT EXPERIMENTAL ORIGIN: Pinus sylvestris
CELL LINE NAME:
CHARACTERISTICS: from 1 to 570 mature peptide sequence
CHARACTERISTICS: Partial sequence for Pinosylvinsynthase from Pinus sylvestris

Claims (18)

1. Pinosylvin synthase genes. 2. Pinosylvinsynthase genes, which thereby characterized records that they are with the plasmid pin 5-49 contained Pinosylvinsynthase-cDNA sequence or its parts or with the cDNA sequence according to SEO ID NO: 1 or its parts hybridize and for Encode pinosylvine synthase. 3. Pinosylvinsynthase genes according to claims 1 and 2, available from dicotyledonous plants. 4. Pinosylvinsynthase genes according to claims 1 to 3, available from Pinus. 5. Regulatory part of the Pinosylvinsyntha se genes according to claims 1 to 4. 6. Structure genes of the Pinosylvinsynthase genes according to Claims 1 to 4. 7. Recombinant prokaryotic or eukaryotic DNA, which one or more Pinosylvinsynthase Ge ne or its parts as claimed in claims 1 to 6 contains "foreign" DNA or as "additional" DNA. 8. Recombinant DNA according to claim 8, which in Pflan cells (including protoplasts) or plants zen (including parts of plants and seeds) hold is.   9. Vectors representing one or more Pinosylvin synthase genes or their parts according to the claims 1 to 8 and / or recombinant DNA according to claim 7 included. 10. Vector plasmid pin 5-49. 11. Transformed microorganisms, which one or several pinosylvine synthase genes or their parts according to claims 1 to 6 and / or recombinant DNA according to claim 7 or vectors according to the An sayings 9 and 10 included. 12. Escherichia coli strain E. coli pin 5-49 and its Mutants. 13. Use of the Pinosylvinsynthase genes and / or its parts and / or the recombinant DNA and / or the vectors and / or the transformed micro organisms according to claims 1 to 12 for trans formation of plant cells (including proto plastic) and plants (including plants divide and seeds). 14. Transformed plant cells (including Pro toplasts) and plants (including plants divide and seeds), which is one or more pino sylvinsynthase genes and / or their parts and / or the recombinant DNA according to claims 1 to 7 contained as "foreign" or "additional" DNA.   15. A method for producing transformed plant cells (including protoplasts) and plants (including plant parts and seeds) with increased ter resistance to pests, characterized in that

• (a) one or more pinosylvine synthase genes or their parts and / or the recombinant DNA according to claims 1 to 7 in the genome of plant cells (including protoplasts) and optionally
• (b) from the transformed plant cells (including protoplasts) completely transformed plants are regenerated and, if appropriate, increased and, if appropriate
• (c) the desired plant parts (including seeds) are obtained from the transformed plants of the parent generation or further generations obtained therefrom.

16. Use of the transformed plant cells (a finally protoplasts) and plants (including Lich plant parts and seeds) according to claim 14 for the production of propagation material and for Generation of new plants that the Pinosylvinsyn thase genes or their parts according to claims 1 to 6 or the recombinant DNA according to claim 7 included and their propagation material.   17. Propagation material available from Ver increased number of transformed plant cells and Plants according to claim 14. 18. Use of DNA sequences which are whole or partially correspond to the cDNA on the plasmid pin 5-49 is included, or in SEO ID NO: 1 leads to the isolation of Pinosylvinsynthase- Genes from plants and in the production of transformed plant cells (including Protoplasts) and plants (including plants divide and seeds).